Lung cancer remains to be the leading cause of cancer related deaths in the United States with poor prognosis. Telomerase, which is upregulated in about 85 percent of lung cancers and not detected in normal lung tissues, is extremely important in cellular proliferation and tumor development. Telomerase elongates telomeres at the end of chromosomes. Thus, it is believed that the modulation of telomerase activity, and telomere length may be a potential therapeutic modality for the treatment of lung cancers. However, molecular mechanisms involved in the regulation of telomerase and telomere length in human lung cancers are not known. The sphingolipid ceramide is known as a tumor suppressor lipid, which mediates anti-proliferative biological responses in human cancer cells. Preliminary results by the PI show that ceramide can regulate telomerase activity, independent of apoptosis or necrosis. Therefore, this proposal focuses on identifying the molecular mechanisms and function of ceramide in the regulation of telomerase activity, and telomere length in human lung cancer cells. Specifically, this proposal aims at: 1) Defining the role of ceramide in the inhibition of telomerase activity in human lung cancer cells. This will be achieved by determining: a) the effects of exogenous C6-ceramide, its biologically inactive analog dihydro-C6-ceramide, and analyzing the effects of overexpression of glucosylceramide synthase, which attenuates ceramide, and b) the effects of increased generation of endogenous ceramide by over-expression of serine palmitoyl transferase, and bacterial sphingomyelinase on telomerase activity in lung cancer cell lines. 2) Determining molecular mechanisms and signaling pathways by which ceramide inhibits telomerase activity in human lung cancer cell lines. This will be done by: a) examining the role of ceramide in the transcription of hTERT (the catalytic subunit of telomerase) via determining its role on the stability of hTERT mRNA, regulation of hTERT promoter activity, and c-Myc protein stability and ubiquitination, and b) determining if ceramide induces the inhibition of telomerase through phosphatase activation, since PP2A which is a downstream target of ceramide has been shown to alter telomerase activity by dephosphorylating hTERT, and 3) Analyzing the mechanisms by which ceramide mediates rapid shortening of telomere length. This will be done by a) analyzing the effects of ceramide on telomere length, b) examining the role of ceramide on the activity of recently identified telomere end-binding proteins, TEF-42 and hPot1, which are potential regulators of telomere length in human lung cancer cells, and c) identification and cloning of TEF-42 protein. The long-term objective of this proposal is to better understand the molecular mechanisms of telomerase regulation, which will then help develop novel and mechanism-driven therapeutic strategies for the treatment of human lung cancers.